Comparison of ribosomal entry and acceptor transfer ribonucleic acid binding sites on Escherichia coli 70S ribosomes. Fluorescence energy transfer measurements from Phe-tRNAPhe to the 3' end of 16S ribonucleic acid

Distances were measured by nonradiative energy transfer from fluorescent probes specifically located on one of three points of yeast or Escherichia coli Phe-tRNAPhe enzymatically bound to the entry site or to the acceptor site of E. coli 70S ribosomes to energy-accepting probes on the 3' end of the 16S ribonucleic acid (RNA) of the 30S subunit. The Y base in the anticodon loop of yeast tRNAPhe was replaced by proflavin. Fluorescein isothiocyanate was attached to the X base (position 47) of E. coli tRNAPhe. E. coli tRNAPhe which had been photochemically cross-linked between positions 8 and 13 followed by chemical reduction to form a fluorescent probe was also used. Labeled tRNAs were aminoacylated and enzymatically bound to the ribosome in the presence of elongation factor Tu and guanosine 5'-triphosphate (acceptor-site binding) or a nonhydrolyzable analogue (entry-site binding). Nonradiative energy transfer measurements were made of the distances between fluorophores located on the Phe-tRNA and the fluorophore at the 3' end of 16S RNA. Calculations were based on comparison of the fluorescence lifetime of the energy donor, located on the Phe-tRNA, in the absence and presence of an energy acceptor on the 3' end of the 16S RNA. Under both sets of binding conditions, the distances to the 3' end of 16S RNA were found to be the following: cross-linked tRNA, greater than 69 A; Y base of tRNA, greater than 61 A. The distance between the 3' end of 16S RNA and the X base of tRNA was found to be 81 A under acceptor-site binding conditions but greater than 86 A under entry-site binding conditions.     

Incorporation of 1,N6-ethenoadenosine into the 3' terminus of tRNA using T4 RNA ligase. 1. Preparation of yeast tRNAPhe derivatives

The 3'-terminal A-C-C-A sequence of yeast tRNAPhe has been modified by replacing either adenosine 76 or 73 with the fluorescent analogues 1,N6-ethenoadenosine (epsilon A) or 2-aza-1,N6-ethenoadenosine (aza-epsilon A). T4 RNA ligase was used to join the nucleoside 3',5'-bisphosphates to the 3' end of the tRNA which was shortened by one [tRNAPhe(-A)] or four [tRNAPhe(-ACCA)] nucleotides. It was found that the base-paired 3'-terminal cytidine 72 in tRNAPhe(-ACCA) is a more efficient acceptor in the ligation reaction than the unpaired cytidine 75 at the A-C-C terminus of tRNAPhe(-A). This finding indicates that the mobility of the accepting nucleoside substantially influences the ligation reaction, the efficiency being higher the lower the mobility. This conclusion is corroborated by the observation that the ligation reaction with the double-stranded substrate exhibits a positive temperature dependence rather than a negative one as found for single-stranded acceptors. The replacement of the 3'-terminal adenosine 76 with epsilon A and aza-epsilon A leads to moderately fluorescent tRNAPhe derivatives, which are inactive in the aminoacylation reaction. A number of other tRNAs (Met, Ser, Glu, Lys and Leu-specific tRNAs both from yeast and Escherichia coli) are also inactivated by epsilon A incorporation. Replacement of adenosine 73 followed by repair of the C-C-A end using nucleotidyl transferase leads to tRNAPhe derivatives which are fully active in the aminoacylation reaction and in polyphenylalanine synthesis. The fluorescence of epsilon A and aza-epsilon A at position 73 is virtually completely quenched, suggesting a stacked arrangement of bases around this position. There is no fluorescence increase when the epsilon A-labeled tRNAPhe is complexed with phenylalanyl-tRNA synthetase, elongation factor Tu, or ribosomes. These observations indicate that the stacked conformation of the 3' terminus is not changed appreciably in these complexes.     

Different conformations of the 3' termini of initiator and elongator transfer ribonucleic acids. An EPR study

The 3' ends of transfer ribonucleic acids were covalently labeled with a nitroxide spin label. The 3' end of initiator tRNA (tRNAMetf) from Escherichia coli shows different motional behavior than the 3' terminus of elongator tRNAs as monitored by EPR. The line shapes of the EPR spectra are quite sensitive to the buffer conditions, as shown by measurements in 4 different buffers. The data are consistent with a constrained or folded back 3' terminus in the initiator tRNA as opposed to the freely rotating elongator 3' terminus. The EPR spectra are also sensitive to aggregation of the tRNA.     

In vitro, the major ribosome binding site on Rous sarcoma virus RNA does not contain the nucleotide sequence coding for the N-terminal amino acids of the gag gene product.

Ribosomes from the reticulocyte lysate bind strongly and mainly to a region located in the 5' end of the Rous sarcoma virus RNA molecule between residues 9 and 53. This binding involves the participation of initiator tRNA and is sensitive to inhibitors of initiation of protein synthesis such as 7-methyl-GMP and aurintricarboxylic acid. The nucleotide sequence of this ribosome binding site has been determined: it conatains a GUG codon centered at position 26 that is not in phase with any termination codon within the 5' end nucleotide sequence of the RNA that we have analyzed (101 residues). However, the predicted N-terminal amino acid sequence starting from this GUG codon (or even from any AUG or GUG codon in the 5' end of the RNA) does not coincide with that of the in vitro-synthesized product of the 5' end proximal gag gene. Nevertheless, inhibition of ribosome binding to this site is accompanied by an inhibition of the in vitro translation of the gag gene.     

The side-by-side model of two tRNA molecules allowing the alpha-helical conformation of the nascent polypeptide during the ribosomal transpeptidation.

Lim and Spirin [25] proposed a preferable conformation of the nascent peptide during the ribosomal transpeptidation. Spirin and Lim [26] excluded the possibilities of the side-by-side model proposed by Johnson et al [13] and the three-tRNA binding model (A, P and E sites) of Rheinberger and Nierhaus [3]. However, a slight conformational change at the 3' end regions of both A and P site tRNA molecules can enable the three different tRNA binding models to converge. With a modification of the angles of the ribose rings of both anticodon and mRNA this model can also be related to the model of Sundaralingam et al [19]. In this model of E coli rRNA the 3' end sequence ACCA76 or GCCA76 of P site tRNA is base-paired to UGGU810 of 23S rRNA, while the ACC75 or GCC75 of A site tRNA are base-paired to GGU1621 23S rRNA. The conformation of the A76 of A site tRNA is necessarily different from that of P site tRNA, at least during the course of the transpeptidation. The A76 of A site tRNA overlaps the binding region of puromycin. The C1400 of 16S rRNA in this model is located at a distance of 4 A from the 5' end of the anticodon of P site tRNA [14] and 17 A from the 5' end of the anticodon of A site tRNA [15]. It is also shown that a considerable but reasonable modification in the conformation of the anticodon loops could lead to accommodation of three deacylated tRNA(Phe) molecules at a time on 70S ribosome in the presence of poly(U) as observed experimentally [6]. A sterochemical explanation for the negatively-linked allosteric interactions between the A and E sites is also shown in the present model.     

Photochemical cross-linking of yeast tRNA(Phe) containing 8-azidoadenosine at positions 73 and 76 to the Escherichia coli ribosome

The 3'-terminal -A-C-C-A sequence of yeast tRNA(Phe) has been modified by replacing either adenosine-73 or adenosine-76 with the photoreactive analogue 8-azidoadenosine (8N3A). The incorporation of 8N3A into tRNA(Phe) was accomplished by ligation of 8-azidoadenosine 3',5'-bisphosphate to the 3' end of tRNA molecules which were shortened by either one or four nucleotides. Replacement of the 3'-terminal A76 with 8N3A completely blocked aminoacylation of the tRNA. In contrast, the replacement of A73 with 8N3A has virtually no effect on the aminoacylation of tRNA(Phe). Neither substitution hindered binding of the modified tRNAs to Escherichia coli ribosomes in the presence of poly(U). Photoreactive tRNA derivatives bound noncovalently to the ribosomal P site were cross-linked to the 50S subunit upon irradiation at 300 nm. Nonaminoacylated tRNA(Phe) containing 8N3A at either position 73 or position 76 cross-linked exclusively to protein L27. When N-acetylphenylalanyl-tRNA(Phe) containing 8N3A at position 73 was bound to the P site and irradiated, 23S rRNA was the main ribosomal component labeled, while smaller amounts of the tRNA were cross-linked to proteins L27 and L2. Differences in the labeling pattern of nonaminoacylated and aminoacylated tRNA(Phe) containing 8N3A in position 73 suggest that the aminoacyl moiety may play an important role in the proper positioning of the 3' end of tRNA in the ribosomal P site. More generally, the results demonstrate the utility of 8N3A-substituted tRNA probes for the specific labeling of ribosomal components at the peptidyltransferase center.     

Ribosome binding by tRNAs with fluorescent labeled 3'' termini.

Yeast and E. coli tRNAPhe samples were oxidized and labeled at the 3' end with dansyl hydrazine or fluorescein thiosemicarbazide. These tRNAs can bind to poly(U)-programmed E. coli 70S tight couple ribosomes in 25 mM magnesium at 8 degrees C. Two binding sites with binding constants of about 1 X 10(9) M-1 (P) and 3 X 10(7) M-1 (A) were determined for the yeast tRNAPhe derivatives. With E. coli tRNAPhe the A site affinity is similar to yeast tRNAPhe but the P site affinity is 5-fold weaker. Singlet-singlet energy transfer showd that the distance from the 3' end of tRNAPhe in the P site to a fluorescein derivative of erythromycin is 23 A. This supports in vitro studies suggesting that erythromycin binds near the peptide moiety of peptidyl tRNA. A distance of 34 A between the 3' ends of 2 tRNAs bound simulatneously on the ribosome was also measured. This long distance may mean that the deacylated fluorescent tRNA binds to the A site in an orientation like that in the stringent response rather than in protein synthesis.     

Domains of the Escherichia coli threonyl-tRNA synthetase translational operator and their relation to threonine tRNA isoacceptors.

The expression of the gene for threonyl-tRNA synthetase (thrS) is negatively autoregulated at the translational level in Escherichia coli. The synthetase binds to a region of the thrS leader mRNA upstream from the ribosomal binding site inhibiting subsequent translation. The leader mRNA consists of four structural domains. The present work shows that mutations in these four domains affect expression and/or regulation in different ways. Domain 1, the 3' end of the leader, contains the ribosomal binding site, which appears not to be essential for synthetase binding. Mutations in this domain probably affect regulation by changing the competition between the ribosome and the synthetase for binding to the leader. Domain 2, 3' from the ribosomal binding site, is a stem and loop with structural similarities to the tRNA(Thr) anticodon arm. In tRNAs the anticodon loop is seven nucleotides long, mutations that increase or decrease the length of the anticodon-like loop of domain 2 from seven nucleotides abolish control. The nucleotides in the second and third positions of the anticodon-like sequence are essential for recognition and the nucleotide in the wobble position is not, again like tRNA(Thr). The effect of mutations in domain 3 indicate that it acts as an articulation between domains 2 and 4. Domain 4 is a stable arm that has similarities to the acceptor arm of tRNA(Thr) and is shown to be necessary for regulation. Based on this mutational analysis and previous footprinting experiments, it appears that domains 2 and 4, those analogous to tRNA(Thr), are involved in binding the synthetase which inhibits translation probably by interfering with ribosome loading at the nearby translation initiation site.     

Specific recognition of the 3'-terminal adenosine of tRNAPhe in the exit site of Escherichia coli ribosomes

Ribosomes from Escherichia coli possess, in addition to A and P sites, a third tRNA binding site, which according to its presumed function in tRNA release during translocation has been termed the exit site. The exit site exhibits a remarkable specificity for deacylated tRNA; charged tRNA, e.g. N-AcPhe-tRNAPhe, is not bound significantly. To determine the molecular basis of this discrimination, we have measured the exit site binding affinities of a number of derivatives of tRNAPhe from E. coli, modified at the 3' end. Binding to the exit site of the tRNAPhe derivatives was measured fluorimetrically by competition with a fluorescent tRNAPhe derivative. We show here that removal of the 2' and 3' hydroxyl groups of the 3'-terminal adenosine decreases the affinity of tRNAPhe for the exit site 15 and 40-fold, respectively. Substitutions at the 3' hydroxyl group (aminoacylation, phosphorylation, cytidylation) as well as removal of the 3'-terminal adenosine (or adenylate) of tRNAPhe lower the affinity below the detection limit of 2 x 10(5) M-1, i.e. more than 100-fold. Modification of the adenine moiety (1,N6-etheno adenine) or replacement of it with other bases (cytosine, guanine) has the same dramatic effect. In contrast, the binding to both P and A sites is virtually unaffected by all of the modifications tested. These results suggest that a major fraction (at least -12 kJ/mol, probably about -17 kJ/mol) of the free energy of exit site binding of tRNAPhe (-42 kJ/mol at 20 mM-Mg2+) is contributed by the binding of the 3'-terminal adenine to the ribosome. The binding most likely entails the formation of hydrogen bonds.     

Evidence for RNA in the peptidyl transferase center of Escherichia coli ribosomes as indicated by fluorescence.

A coumarin derivative was covalently attached to either the amino acid or the 5' end of phenylalanine-specific transfer RNA (tRNA(phe)). Its fluorescence was quenched by methyl viologen when the tRNA was free in solution or bound to Escherichia coli ribosomes. Methyl viologen as a cation in solution has a strong affinity for the ionized phosphates of a nucleic acid and so can be used to qualitatively measure the presence of RNA in the immediate vicinity of the tRNA-linked coumarins upon binding to ribosomes. Fluorescence lifetime measurements indicate that the increase in fluorescence quenching observed when the tRNAs are bound into the peptidyl site of ribosomes is due to static quenching by methyl viologen bound to RNA in the immediate vicinity of the fluorophore. The data lead to the conclusion that the ribosome peptidyl transferase center is rich in ribosomal RNA. Movement of the fluorophore at the N-terminus of the nascent peptide as it is extended or movement of the tRNA acceptor stem away from the peptidyl transferase center during peptide bond formation appears to result in movement of the probe into a region containing less rRNA.     

Aminoacylation complex structures of leucyl-tRNA synthetase and tRNALeu reveal two modes of discriminator-base recognition

Leucyl-tRNA synthetase (LeuRS) specifically recognizes the characteristic long variable arm and the discriminator base, A73, of tRNA(Leu) in archaea and eukarya. The LeuRS 'editing domain' hydrolyzes misformed noncognate aminoacyl-tRNA. Here we report the crystal structure of the archaeal Pyrococcus horikoshii LeuRS-tRNA(Leu) complex. The protruding C-terminal domain of LeuRS specifically recognizes the bases at the tip of the long variable arm. The editing domain swings from its tRNA-free position to avoid clashing with the tRNA. Consequently the tRNA CCA end can bend and reach the aminoacylation active site. The tRNA 3' region assumes two distinct conformations that allow A73 to be specifically recognized in different ways. One conformation is the canonical 'aminoacylation state.' The other conformation seems to be the 'intermediate state,' where the misaminoacylated 3' end has partially relocated to the editing domain.     

Syntheses and properties of fluorescent labeled oligonucleotides containing deoxyethenoadenosine at 5' end.

The fluorescent labeled oligodeoxyribonucleotides which contain deoxyethenoadenosione (d epsilon A) at their 5' end were prepared by treating CPG bound oligonucleotides with 5'-DMTr-deoxyethenoadenosine-3'-H-phosphonate. The hybrid formation of d epsilon A-oligonucleotide with its complementary DNA was studied by fluorescence spectroscopy. The fluorescence of d epsilon A in a single strand was largely quenched by stacking interaction with the base at 3' position. When d epsilon A-oligonucleotides hybridized with their complementary strands, relative fluorescence quantum yields (Qrel) against d epsilon A changed in specific manners. These results suggest that d epsilon A-oligonucleotides are applicable to study the local structure of DNA in solution.     

Binding of the 3'' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA.     

A fluorescent derivative of ribosomal protein S18 which permits direct observation of messenger RNA binding.

70 S Escherichia coli ribosomes were reacted with the fluorescent dye N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid for 10 min under mild conditions. The resulting ribosomes were fully active. 30 S subunits isolated from these particles were also fully active. They contain approximately 0.7 eq of fluorescent dye. Nearly all of it is attached to protein S18. Competitive reaction with N-ethylmaleimide implies that the fluorescent dye is located at cysteine 10 of the protein. The labeled 30 S particles will recombine with 50 S subunits to form stable 70 S particles. Thus the procedures we have developed allow the large scale preparation of an active fluorescent conjugate of the 70 S ribosome. The fluorescence of the 70 S particles is sensitive to the binding of mRNA, showing both quenching and a shift in emission spectra. Thus it affords a simple way to quantitate mRNA binding directly. In pilot studies without tRNA, the binding constant of the initiation triplet codon adenylyl-(3' leads to 5')-uridylyl-(3' leads to 5')-guanosine to 70 S ribosome was found to be an order of magnitude larger than that of polyuridylic acid.     

Orientations of transfer RNA in the ribosomal A and P sites.

In protein synthesis, peptide bond formation requires that the tRNA carrying the amino acid (A site tRNA) contact the tRNA carrying the growing peptide chain (P site tRNA) at their 3' termini. Two models have been proposed for the orientations of two tRNAs as they would be bound to the mRNA in the ribosome. Viewing the tRNA as an upside down L, anticodon loop pointing down, acceptor stem pointing right, and calling this the front view, the R (Rich) model would have the back of the P site tRNA facing the front of the A site tRNA. In the S (Sundaralingam) model the front of the P site tRNA faces the back of the A site tRNA. Models of two tRNAs bound to mRNA as they would be positioned in the ribosomal A and P sites have been created using MC-SYM, a constraint satisfaction search program designed to build nucleic acid structures. The models incorporate information from fluorescence energy transfer experiments and chemical crosslinks. The models that best answer the constraints are of the S variety, with no R conformations produced consistent with the constraints.     

Substrate-assisted catalysis of peptide bond formation by the ribosome

The ribosome accelerates the rate of peptide bond formation by at least 10(7)-fold, but the catalytic mechanism remains controversial. Here we report evidence that a functional group on one of the tRNA substrates plays an essential catalytic role in the reaction. Substitution of the P-site tRNA A76 2' OH with 2' H or 2' F results in at least a 10(6)-fold reduction in the rate of peptide bond formation, but does not affect binding of the modified substrates. Such substrate-assisted catalysis is relatively uncommon among modern protein enzymes, but it is a property predicted to be essential for the evolution of enzymatic function. These results suggest that substrate assistance has been retained as a catalytic strategy during the evolution of the prebiotic peptidyl transferase center into the modern ribosome.     

Rapid kinetic analysis of EF-G-dependent mRNA translocation in the ribosome

Precise and coordinated movement of the tRNA-mRNA complex within the ribosome is a fundamental step during protein biosynthesis. The molecular mechanism for this process is still poorly understood. Here we describe a new sensitive method for monitoring elongation factor G-dependent translocation of the mRNA in the ribosome. In this method, the fluorescent probe pyrene is covalently attached to the 3' end of a short mRNA sequence at position +9. Translocation of the mRNA by one codon results in a significant decrease in the fluorescence emission of pyrene and can be used to directly monitor mRNA movement using rapid kinetic methods. Importantly, this method offers the flexibility of using any tRNA or tRNA analog in order to elucidate the molecular mechanism of translocation. Our results show that the mRNA is translocated at the same rate as the tRNAs, which is consistent with the view that the movement of the tRNAs and the mRNA are coupled in the ribosome. Furthermore, an anticodon stem-loop analog of tRNA is translocated from the ribosomal A site at a rate constant that is 350-fold lower than peptidyl tRNA, indicating that the D stem, T stem and acceptor stem of A site tRNA contribute significantly to the rate of translocation.     

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

The many interactions of tRNA with the ribosome are fundamental to protein synthesis. During the peptidyl transferase reaction, the acceptor ends of the aminoacyl and peptidyl tRNAs must be in close proximity to allow peptide bond formation, and their respective anticodons must base pair simultaneously with adjacent trinucleotide codons on the mRNA. The two tRNAs in this state can be arranged in two nonequivalent general configurations called the R and S orientations, many versions of which have been proposed for the geometry of tRNAs in the ribosome. Here, we report the combined use of computational analysis and tethered hydroxyl-radical probing to constrain their arrangement. We used Fe(II) tethered to the 5' end of anticodon stem-loop analogs (ASLs) of tRNA and to the 5' end of deacylated tRNA(Phe) to generate hydroxyl radicals that probe proximal positions in the backbone of adjacent tRNAs in the 70S ribosome. We inferred probe-target distances from the resulting RNA strand cleavage intensities and used these to calculate the mutual arrangement of A-site and P-site tRNAs in the ribosome, using three different structure estimation algorithms. The two tRNAs are constrained to the S configuration with an angle of about 45 degrees between the respective planes of the molecules. The terminal phosphates of 3'CCA are separated by 23 A when using the tRNA crystal conformations, and the anticodon arms of the two tRNAs are sufficiently close to interact with adjacent codons in mRNA.